Traumatic brain injury (TBI) results in the prolonged activation of calpains, which contributes to cytoskeletal damage, neuronal death and behavioral dysfunction. However, surprisingly few in vivo cellular substrates of calpains have been identified in the traumatically injured brain and, consequently, little is understood about the pathways through which calpains mediate posttraumatic morbidity. Calpastatin, the endogenous inhibitor of calpains, is the only known protein that exclusively inhibits calpains. As such, calpastatin represents an ideal molecular tool with which to isolate the actions of calpains within the injured brain. Efforts to translate exciting preclinical data demonstrating functional improvement in brain-injured rodents treated with exogenous calpain inhibitors have been slowed by challenges with solubility, specificity and bioavailability of small molecule inhibitors. Enhancing endogenous calpastatin activity may represent a novel and potent therapeutic approach. The overall goals of Project 1, then, are to evaluate the role of calpastatin in regulating posttraumatic calpain-mediated proteolysis and to assess the neuroprotective and behavioral efficacy of increasing calpastatin activity in the setting of TBI. Using genetically altered mice that either overexpress human calpastatin or are calpastatin deficient, Project 1 will: 1) evaluate the role of calpastatin in modulating behavioral outcome following focal or diffuse brain injury, 2) quantify the effects of altered calpastatin expression on neuronal survival and axonal injury after focal or diffuse brain injury, 3) determine the in vivo role of calpastatin in limiting trauma-induced proteolysis of neuronal cytoskeletal proteins, and 4) evaluate the role of posttraumatic calpain activation in modifying membrane proteins involved in calcium influx and in modulating mitochondria-related cell death events. Our central hypothesis is that calpastatin overexpression will prevent calpain-mediated cleavage of neuronal substrates critical for cell survival, thereby attenuating posttraumatic neuronal death and dysfunction. The proposed experiments will provide the first evidence for a functional role for calpastatin in posttraumatic pathology and elucidate differential roles for the calpain/calpastatin system in focal and diffuse TBI. In addition, this Project will provide the groundwork for novel therapeutic approaches, based on manipulation of the calpastatin system, aimed at attenuating brain damage and dysfunction due to TBI as well as other CMS injury and disease states.